PROJECT SUMMARY Acute chest syndrome (ACS), a type of acute lung injury, is one of the leading causes of mortality in Sickle Cell Disease (SCD). Current treatments for ACS are primarily supportive, and there is a critical need for rescue therapies that can halt the progression of ACS. ACS is often a sequela of acute systemic vaso-occlusive crisis and preceded by thrombocytopenia. However, the role of platelets in the pathogenesis of ACS remains largely unknown. We hypothesize that ACS involves NLRP3-inflammasome mediated release of IL-1?-carrying platelet exosomes in SCD, which promote platelet-neutrophil aggregation leading to arrest of blood flow in lung. We also propose that targeted inhibition of TLR4/NLRP3-caspase-1 signaling in platelets is a potential therapy for ACS. To test this hypothesis, we will use an integrative physiologic approach that utilizes our recently validated model of lipopolysaccharide (LPS) induced vaso-occlusive crisis in transgenic SCD mice, in vivo multi-photon excitation (MPE) imaging of the lung vasculature in live SCD mice, live cell imaging of SCD human blood flowing in microfluidic channels in vitro, SCD mice lacking caspase-1 in platelets and nanoparticle tracking analyses of exosomes. In Aim 1, we will determine whether release of IL-1?-carrying platelet exosomes in SCD promote platelet-neutrophil aggregates in pulmonary arterioles that result in loss of blood flow in the lung. In Aim 2, we will determine whether TLR4/NLRP3-inflammasome mediated activation of caspase-1 in platelets is responsible for release of IL-1?-carrying exosomes from platelets in SCD. We have developed platelet-targeted nanomedicine (PTN) that specifically recruits to sites of platelet aggregation and thrombosis in vivo for targeted drug delivery. In Aim 3, we will determine whether PTNs carrying TLR4 or caspase-1 inhibitors, or IL-1 receptor antagonist (IL-1RA) can selectively recruit to sites of platelet aggregation to block platelet exosome release and signaling, and stop platelet-neutrophil aggregation in the lung vasculature of SCD mice. This study will identify a novel, platelet-derived exosome-mediated mechanism contributing to ACS. The findings will also establish that delivery of TLR4 or caspase-1 inhibitor, or IL-1RA encapsulated in PTNs can be a potential rescue therapy for ACS in high risk SCD patients presenting with vaso-occlusive crisis and thrombocytopenia.